P21 waf1 cip1 12d1 rabbit mab

Manufactured by Cell Signaling Technology

Sourced in China

P21 Waf1/Cip1 (12D1) rabbit mAb is a monoclonal antibody that recognizes the p21 Waf1/Cip1 protein. p21 Waf1/Cip1 is a cyclin-dependent kinase inhibitor that plays a role in cell cycle regulation.

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5 protocols using p21 waf1 cip1 12d1 rabbit mab

Antibodies for Cell Cycle Analysis

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We used the following antibodies:

p53 (7F5) rabbit monoclonal antibody (mAb), 2527 (1:500; Cell Signaling Technology, Danvers, MA)

p21 Waf1/Cip1 (12D1) rabbit mAb, 2947 (1:1000; Cell Signaling Technology)

Phospho-Rb (Ser-807/811; D20B12) rabbit mAb, 8516 (1:500; Cell Signaling Technology)

GAPDH (D16H11) rabbit mAb, 5174 (1:1000; Cell Signaling Technology)

Cyclin D2 (D52F9) rabbit mAb, 3741 (1:500; Cell Signaling Technology)

Anti-centrin, clone 20H5, 04-1624 (1:500; EMD Millipore)

Anti–α-tubulin antibody, ab15246 (1:500; Abcam, Cambridge, UK)

Phospho–histone H2AX (Ser-139; 20E3) rabbit mAb, 9718 (1:500; Cell Signaling Technology)

Anti–β-actin antibody, ab8227 (1:5000; Abcam)

Secondary antibodies for immunofluorescence (Alexa 488 and Alexa 594 conjugates) were obtained from Life Technologies and used at 1:500 dilution. Secondary HRP-conjugated antibodies for Western blotting were from Cell Signaling Technology and typically used at 1:5000 dilution.

Potapova T.A., Seidel C.W., Box A.C., Rancati G, & Li R. (2016). Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53. Molecular Biology of the Cell, 27(20), 3065-3084.

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Comprehensive Cellular Assay Protocol

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The source of materials adopted in this article
can be found in our previous reported article.^{11 (link)} In addition, the flow cytometry (FCM) anti-P-glycoprotein
antibody and mouse IgG2a isotype control were supplied by Abcam (Cambridge,
UK). The anti-P-glycoprotein antibody, anti-cyclin A2 antibody, anti-β
actin antibody, and anti-β tubulin antibody were offered by
Abcam. The anti-cyclin D1 antibody was supplied by Beyotime (Shanghai,
China). P21 Waf1/Cip1 (12D1) rabbit mAb, the caspase-9 antibody, the
caspase-3 antibody, LC3A/B rabbit mAb, the Beclin-1 rabbit mAb, and
Atg12 rabbit mAb were obtained from the Cell Signaling Technology,
Inc. (Shanghai, China). All other chemicals unless otherwise indicated
were of analytical grade from Alladin Co. Ltd (Shanghai, China).

Chen S.Q., Wang C., Song Y.Q., Tao S., Yu F.Y., Lou H.Y., Hu F.Q, & Yuan H. (2020). Quercetin Covalently Linked Lipid Nanoparticles: Multifaceted Killing Effect on Tumor Cells. ACS Omega, 5(46), 30274-30281.

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Apoptosis Signaling Pathway Inhibition

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Cladribine (Sigma Co., St. Louis, MO) and entinostat (LC Laboratories, Inc., Woburn, MA) were dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at 250 mmol/L and 200 mmol/L, respectively. The stock solutions were stored at −20°C.
The sources of antibodies for western blot assays were as follows: caspase-3 rabbit mAb (8G10), caspase-8 (1C12) mouse mAb, caspase-9 (Asp353) rabbit mAb, PARP rabbit mAb, P-Histone H2A.X (Ser139) rabbit antibody, Acetyl-Histone H3 (Lys9), Histone H3, P-CHK1 (Ser345) (133D3) rabbit mAb, CHK1 rabbit antibody, P-CHK2 (Thr68) rabbit polyclonal antibody, CHK2 rabbit polyclonal antibody and p21^Waf1/Cip1 (12D1) rabbit mAb (Cell Signaling Technology, Inc., Beverly, MA); Cyclin D1 rabbit mAb, E2F-1 mouse mAb (KH95), p27 (F-8) mouse mAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA); β-actin mouse mAb (clone AC-75) (Sigma Co.). All other reagents were purchased from Sigma Co. unless otherwise specified.

Wang B., Lyu H., Pei S., Song D., Ni J, & Liu B. (2018). Cladribine in combination with entinostat synergistically elicits anti-proliferative/anti-survival effects on multiple myeloma cells. Cell Cycle, 17(8), 985-996.

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Total Protein Extraction and Western Blot

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To prepare total protein, cells were extracted with lysis buffer containing 20 mM Hepes- KOH, pH 7.4, 120 mM NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, a cocktail of protease inhibitors (Complete^TM, Roche Applied Science, Laval QC) and a cocktail of phosphatase inhibitors (PhosStop^TM, Roche Applied Science). The antibodies (Abs) in this study were Bcl-xL (54H6) rabbit monoclonal Ab (mAb), Ki-67(8D5) mouse mAb, p21Waf1/Cip1(12D1) rabbit mAb, p16/INK4A rabbit polyclonal Ab (pAb) and p53(1C12) mouse mAb obtained from Cell Signaling Technology Inc. (Beverly, MA). Phospho-histone H2A.X (Ser139) (JBW301) mouse mAb were purchased from EMD Millipore Corporation (Temecula, CA), and β-actin (AC-15) mouse mAb was from Abcam Inc. (Cambridge, MA). Peroxidase-labeled secondary Ab were detected by enhanced chemiluminescence with reagent set from GE Healthcare Life Science (Mississauga, ON) or SuperSignal WestPico chemiluminescence substrates from Thermo Scientific (Rockford, IL).

Baruah P.S., Beauchemin M., Hébert J, & Bertrand R. (2016). Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts. PLoS ONE, 11(7), e0159091.

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Protein Expression Analysis of Myometrial and Leiomyoma Cells

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Protein lysates were extracted from myometrial and leiomyoma cells using RIPA lysis and extraction buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific). The protein concentration was determined using BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of proteins were subjected to SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using the following primary antibodies: p21 Waf1/Cip1 (12D1) Rabbit mAb (Cell Signaling Technology), Human p16INK4a/CDKN2A Antibody (Fisher Scientific), pAKT and AKT (Cell Signaling Technology), HMGA2 (Biocheck Inc), and β-actin Antibody (Cell Signaling Technology). Secondary antibodies were horseradish peroxidase (HRP)-labeled anti-mouse (7076S, Cell Signaling Technology), anti-rabbit (7074S, Cell Signaling Technology), or anti-goat (HAF109, Fisher Scientific). Chemiluminescence was detected by adding a chemiluminescent HRP substrate (Thermo Fisher Scientific) and measured with a Fujifilm LAS-3000 Imager.

Xie J., Ubango J., Ban Y., Chakravarti D., Kim J.J, & Wei J.J. (2018). Comparative Analysis of AKT and the related biomarkers in uterine leiomyomas with MED12, HMGA2 and FH mutations. Genes, chromosomes & cancer, 57(10), 485-494.

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